Chip based DNA microarrays are an integration of circuit fabrication technology and genetics. DNA microarrays consist of matrices of DNA arranged on a solid surface where the DNA at each position recognizes the expression of a different target sequence. Microarrays are used to identify which genes are turned on or off in a cell or tissue, and to evaluate the activity level under various conditions. This knowledge enables researchers to determine whether a cell is diseased or the effect of a drug on a cell or group of cells. These studies are critical to determine a drug""s efficacy or toxicity, to identify new drug targets, and to more accurately diagnose illnesses, such as specific types of cancer. The technology is useful to classify tumors with the hope of establishing a correlation between a specific type of cancer, the therapeutic regiment used for treatment, and survival.
Photolithography technology, similar to that employed for transistor etching into silicon chips, is often used to layer chains of nucleotides, the basic units of DNA, onto silicon. Additionally, nucleotides, often referred to as xe2x80x9cprobes,xe2x80x9d may be deposited onto solid substrates, or solid substrates coated with various polymers. Various deposition or spraying methods are used to deposit the nucleotides, including piezoelectric technology similar to that used for ink-jet printer heads and robotic methods. The probes are attached to the substrates or polymers by thermal, chemical, or light-based methods to form the microarray.
The genes of interest, or xe2x80x9ctargets,xe2x80x9d are generally put into solution in a xe2x80x9cfluidics stationxe2x80x9d which disperses the target solution on the microarray surface. If fluorescence detection is to be used, the targets may be tagged with fluorescent labels. Nucleotide targets which are complementing, or xe2x80x9crecognizedxe2x80x9d by, the nucleotide probes on the support or polymer then bind, or hybridize, with their corresponding probes. Additionally, the targets may be enzymatically tagged after hybridization to their respective probes. After rinsing to remove any unbound targets from the microarray, the presence and or concentration of specific targets is determined by spectroscopic or other methods.
Many beneficial applications exist for microarrays, including diagnosing mutations in HIV-1, studying the gene defects which lead to cancer, polymorphism screening and genotyping, and isolating the genes which lead to genetic based disorders, such as multiple sclerosis.
A microarray is generally formed by coating a solid support with a polymer. Acrylamide (CH2=CHC(O)NH2; C.A.S. 79-06-1; also known as acrylamide monomer, acrylic amide, propenamide, and 2-propenamide) is an odorless, free-flowing white crystalline substance that is used as a chemical intermediate in the production and synthesis of polyacrylamide polymers. Polyacrylamides have a variety of uses and can be modified to optimize nonionic, anionic, or cationic properties for specified uses, such as a polymer coating for the solid support of a microarray.
Polyacrylamide hydrogels are often used as molecular sieves for the separation of nucleic acids, proteins, and other moieties, and as binding layers to adhere to the surfaces biological molecules including, but not limited to, proteins, peptides, oligonucleotides, polynucleotides, and larger nucleic acid fragments. The gels currently are produced as thin sheets or slabs, typically by depositing a solution of acrylamide monomer, a crosslinker such methylene bisacrylamide, and an initiator such as N, N, Nxe2x80x2, Nxe2x80x2-tetramethylethylendiamine (TEMED) between two glass surfaces, such as microscope slides. A spacer is used to obtain the desired thickness of polyacrylamide.
Generally, the acrylamide polymerization solution is a 4-5% solution (acrylamide/bisacrylamide 19/1) in water/glycerol, with a nominal amount of initiator added. The solution is polymerized and crosslinked either by ultraviolet (UV) radiation (e.g., 254 nm for at least about 15 minutes, or other appropriate UV conditions, collectively termed xe2x80x9cphotopolymerizationxe2x80x9d), or by thermal initiation at elevated temperature, typically about 40xc2x0 C. Following polymerization and crosslinking, the top glass slide is removed from the surface to uncover the gel. The pore size (or xe2x80x9csieving propertiesxe2x80x9d) of the gel is controlled by changing the amount of crosslinker and the % solids in the monomer solution. The pore size also can be controlled by changing the polymerization temperature.
In the fabrication of polyacrylamide hydrogel arrays used as binding layers for biological molecules, the acrylamide solution typically is imaged through a mask during the UV polymerization/crosslinking step. The top glass slide is removed after polymerization, and the unpolymerized monomer is washed away with water leaving a fine feature pattern of polyacrylamide hydrogel, the crosslinked polyacrylamide hydrogel pads.
Further, in an application of lithographic techniques known in the semiconductor industry, light can be applied to discrete locations on the surface of a polyacrylamide hydrogel to activate these specified regions for the attachment of an anti-ligand, such as an antibody or antigen, hormone or hormone receptor, oligonucleotide, or polysaccharide, to the hydrogel (PCT International Application WO 91/07087, incorporated by reference). Following fabrication of the hydrogel array, the polyacrylamide subsequently is modified to include functional groups for the attachment of probes. The probes, such as DNA, are later attached.
Chemical immobilization of biomolecules, such as DNA, RNA, peptides, and proteins, on a solid support or within a matrix material, such as a hydrogel, has become a very important aspect of molecular biology research. This is especially true in the manufacturing and application of microarray or chip-based technologies where biomolecules are immobilized as probes.
Typical procedures for attaching a biomolecule to a surface involve multiple reaction steps, often requiring chemical modification of the hydrogel to provide the chemical functionality for covalent bonding with the biomolecule. The efficiency of the attachment chemistry and strength of the chemical bonds formed are critical to the fabrication and ultimate performance of the microarray.
For polyacrylamide, the necessary functionality for probe attachment presently entails chemical modification of the hydrogel through the formation of amide, ester, or disulfide bonds after polymerization and crosslinking of the hydrogel. An unresolved problem with this approach is the less than optimal stability of the attachment chemistry over time, especially during subsequent manufacturing steps, and under use conditions where the microarray is exposed to high temperatures, ionic solutions, and multiple wash steps. Such conditions promote continued depletion in the quantity of probe molecules present in the array, thus reducing its performance and useful life. A further problem is the low efficiency of the method.
Another approach that has been employed is the polymerization of a suitable xe2x80x9cattachment co-monomerxe2x80x9d into the polyacrylamide matrix that is capable of bonding with the DNA oligonucleotide probe. However, this method is limited in that the incorporation of the attachment co-monomer as a third component of the matrix, along with the acrylamide monomer and crosslinker, can give rise to problems during acrylamide polymerization. These problems include an inability to form the matrix, a loss of mechanical integrity in the matrix, and a loss of adhesion between the matrix and the solid support.
A more recent method has employed direct co-polymerization of an acrylamide-derivatized oligonucleotide. For instance, ACRYDITE (Mosaic Technologies, Boston, Mass.) is an acrylamide phosphoramidite that contains an ethylene group capable of free radical polymerization with acrylamide. Acrydite-modified oligonucleotides are mixed with acrylamide solutions and polymerized directly into the gel matrix (Rehman et al., Nucleic Acids Research, 27, 649-655 (1999). This method still relies on acrylamide as the monomer. Depending on the choice of chemical functionality, similar problems in the stability of attachment, as with the above-mentioned methods, also result.
Accordingly, the prior art methods use post-modification of the matrix, or incorporation of a suitable co-monomer during the fabrication process. In addition to the disadvantages described above, toxic acrylamide monomer is used in manufacturing the arrays.
The present invention seeks to overcome some of the aforesaid disadvantages of the prior art, including the problems associated with chemical attachment of the probes to the polymer-coated support, for the purpose of forming microarrays useful in expression and single nucleotide polymorphism (SNP) analysis. In particular, the present invention provides methods of performing expression and SNP microarray analysis to determine the presence and/or concentration of a target, wherein a microarray is formed by attaching a polymer-coated support and a probe by a [2+2] cycloaddition reaction, wherein the reaction is between reactive sites on the polymer and probe. These and other objects and advantages of the present invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
Novel hydrogel arrays are used to detect specific target oligonucleotides, including mRNA and DNA. Expression and single nucleotide polymorphism analyses are performed. The arrays are constructed from polyacrylamide based hydrogels and synthetic oligonucleotide probes that are functionalized with reactive groups. The reactive groups undergo [2+2] type photocycloaddition when exposed to ultraviolet light. This cycloaddition results in the probes being covalently attached to the hydrogel.